Human neuronal attachment factor-1

ABSTRACT

A human F-spondin-like protein and DNA (RNA) encoding such protein and a procedure for producing such protein by recombinant techniques is disclosed. Also disclosed are methods for utilizing such polypeptide for treating spinal cord injuries and damage to peripheral nerves by promoting neural-cell adhesion and neurite extension, inhibiting tumor metastases and tumor angiogenesis, and stimulating wound repair. Antagonists are also disclosed which may be utilized to prevent malaria. Diagnostic assays for identifying mutations in nucleic acid sequence encoding a polypeptide of the present invention and for detecting altered levels of the polypeptide of the present invention for detecting diseases, for example, cancer, are also disclosed.

This application is a division application Ser. No. 08/799,173, filed onFeb. 12, 1997, now U.S. Pat. No. 5,871,969 which claims benefit of U.S.Provisional Application Serial No. 60/011,519 filed Feb. 12, 1996.

This invention relates to newly identified polynucleotides, polypeptidesencoded by such polynucleotides, the use of such polynucleotides andpolypeptides, as well as the production of such polynucleotides andpolypeptides. More particularly, the polypeptide of the presentinvention has been putatively identified as a human neuronal attachmentfactor-1, sometimes hereinafter referred to as “NAF-1”. The inventionalso relates to inhibiting the action of such polypeptides.

BACKGROUND OF THE INVENTION

F-spondin (FSP) is a gene that is predominantly expressed during theearly development of the vertebrate nervous system. The main function isthought to be in neural cell pattern formation and axonal growth. It wasfound in a subtractive hybridization screen designed to isolatefloor-plate specific genes. The floor-plate provides diffusible signalsthat act on the neurons that extend from the developing spinal cord.These signals can lead to chemoattraction and fasciculation ofcommissural axons in the ventral midline. F-spondin mRNA is expressed athigh levels in the developing neural tube at the ventral midline evenbefore cell differentiation markers can detect the floor-plate.F-spondin is not detectable in other regions of the spinal cord untillater in embryonic life. There is also transient F-spondin expressionearly in peripheral nerve development which diminishes to undetectablelevels following birth. The adult central nervous system containsF-spondin while the peripheral nerve (sciatic nerve) does not. Outsidethe adult nervous system, organs such as the lung and kidney alsoexpress F-spondin. The protein is 807 anmino acids and codes for apredicted 90 kD polypeptide. The apparent size is approximately 116 kDby SDS-PAGE which indicates post-translational modifications such asglycosylation. There are six domains homologous to the thrombospondin(TSP) type 1 repeats (TSR) which have been shown to control celladhesion. The protein has been expressed in COS cells and purified as amyc-tag fusion protein. This protein was active in promoting neuriteextension and adhesion of embryonic dorsal root ganglion and dorsalspinal cords respectively. It was not chemotropic for embryonic dorsalspinal cord neurons. (Klar, A. et al., Cell, 69:95-110 (1992)).

The C-terminal half of F-spondin contains 6 repeats identified inthrombospondin and other proteins implicated in cell adhesion.Thrombospondin is a 450,000-dalton glyco-protein secreted by plateletsin response to such physiological activators as thrombin and collagen(Lawler, J., Blood, 67:1197-1209 (1986)). TSP comprises 3% of the totalplatelet protein and 25% of the total platelet-secreted proteins(Tuszynslci, G. P., et al., J. Biol. Chem., 260:12240-12245 (1985)).Although the precise biological role of TSP has yet to be fullyestablished, it is generally accepted that TSP plays a major role incell adhesion and cell-cell interactions. It should be pointed out thatthe C-terminal repeats present in thrombospondin may have differentbiological activities.

TSP was found to promote the cell-substratum adhesion of a variety ofcells, including platelets, melanoma cells, smooth muscle cells,endothelial cells, fibroblasts and epithelial cells (Tuszynski, G. P.,et al., Science (Washington, D.C.), 236:1570-1573 (1983)).

Thrombospondin has been postulated to play a role in malarial infectioninduced by only one strain of malaria, plasmodium falciparum. Duringmalarial infection, TSP promotes adhesion of parasitized red cells toendothelial cells (Roberts, D. D., et al., Nature (Lond.), 318:64-66(1984)) and during tumor cell metastases TSP promotes adhesion of mousesarcoma cells to the vascular bed and expression of the malignantphenotype of small cell carcinoma (Castle, V. J., J. Clin. Invest.,87:1883-1883 (1991)).

Properdin is a complement-binding protein which also contains the 6terminal repeats found in thrombospondin. UNC-5, a C. elegans gene thatbears two terminal repeats, appears to guide the axonal extension of thesub-set of neurons. These proteins, which contain at least one member ofthe six terminal repeats, form a family of proteins which have relatedfunctions.

The gene and polypeptide encoded thereby of the present invention hasbeen putatively identified as an Neuronal Attachment Factor-1 protein asa result of amino acid sequence homology to rat F-spondin.

SUMMARY OF THE INVENTION

In accordance with one aspect of the present invention, there isprovided a novel mature polypeptide, as well as biologically active anddiagnostically or therapeutically useful fragments, analogs andderivatives thereof. The polypeptide of the present invention is ofhuman origin.

In accordance with another aspect of the present invention, there areprovided isolated nucleic acid molecules encoding a polypeptide of thepresent invention including mRNAs, cDNAs, genomic DNAs as well asanalogs and biologically active and diagnostically or therapeuticallyuseful fragments thereof.

In accordance with another aspect of the present invention there isprovided an isolated nucleic acid molecule encoding a mature polypeptideexpressed by the human cDNA contained in ATCC Deposit No. 97343.

In accordance with yet a further aspect of the present invention, thereis provided a process for producing such polypeptide by recombinanttechniques comprising culturing recombinant prokaryotic and/oreukaryotic host cells, containing a nucleic acid sequence encoding apolypeptide of the present invention, under conditions promotingexpression of said protein and subsequent recovery of said protein.

In accordance with yet a further aspect of the presentcinvention, thereis provided a process for utilizing such polypeptide, or polynucleotideencoding such polypeptide for therapeutic purposes, for example, totreat spinal cord injuries or damage to peripheral nerves by promotingneural cell adhesion and neurite extension, to inhibit tumor cellmetastases, inhibit endothelial cell proliferation, adhesion andmotility, to decrease tumor neovascularization, to be angiostatic fortumor cells and to promote wound healing.

In accordance with yet a further aspect of the present invention, thereare provided antibodies against such polypeptides, which would bind toand neutralize NAF-1 to inhibit its putative cell adhesion properties torestrict metastases, particularly tumor metastases.

In accordance with another aspect of the present invention, there areprovided NAF-1 agonists which mimic NAF-1 and binds to the NAF-1receptors.

In accordance with yet another aspect of the present invention, thereare provided antagonists to such polypeptides, which may be used toinhibit the action of such polypeptides, for example, in the treatmentof malarial infection caused by Plasmodium falciparum.

In accordance with yet a further aspect of the present invention, thereis also provided nucleic acid probes comprising nucleic acid moleculesof sufficient length to hybridize to a nucleic acid sequence of thepresent invention.

In accordance with still another aspect of the present invention, thereare provided diagnostic assays for detecting diseases or susceptibilityto diseases related to mutations in the nucleic acid sequences encodinga polypeptide of the present invention.

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing such polypeptides, orpolynucleotides encoding such polypeptides, for in vitro purposesrelated to scientific research, for example, synthesis of DNA andmanufacture of DNA vectors.

These and other aspects of the present invention should be apparent tothose skilled in the art from the teachings herein.

BRIEF DESCRIPTION OF THE FIGURES

The following drawings are illustrative of embodiments of the inventionand are not meant to limit the scope of the invention as encompassed bythe claims.

FIG. 1 is an illustration of the cDNA (SEQ ID NO:1) and correspondingamino acid sequence (SEQ ID NO:2) of the polypeptide of the presentinvention. Sequencing was performed using a 373 automated DNA sequencer(Applied Biosystems, Inc.). The putative leader sequence region isunderlined.

FIG. 2 is an amino acid sequence comparison between the polypeptide ofthe present invention (bottom line) (SEQ ID NO:2) and rat F-spondin(rFSP) (top line) (SEQ ID NO:7).

FIG. 3 is an amino acid sequence comparison between the cell adhesionsequence of NAF-1 (FLP-TSR; SEQ ID NO:19) and the six cell adhesionsequences of rat F-spondin (FSR-TSR-1, -2, -3, -4, -5, and -6; SEQ IDNOS:8-13, respectively). Also shown is a TSR consensus sequences shownin the sequence listing as SEQ ID NO:14.

FIG. 4 shows an analysis of the NAF-1 amino acid sequence (SEQ ID NO:2).Alpha, beta, turn and coil regions; hydrophilicity and hydrophoicity;amphipathic regions; flexible regions; antigenic index and surfaceprobability are shown. In the “Antigenic Index-Jameson-Wolf” graph, thepositive peaks indicate locations of the highly antigenic regions of theNAF-1 protein, i.e., regions from which epitope-bearing peptides of theinvention can be obtained.

DETAILED DESCRIPTION

In accordance with an aspect of the present invention, there is providedan isolated nucleic acid (polynucleotide) which encodes for the maturepolypeptide having the deduced amino acid sequence of FIG. 1 (SEQ IDNO:2).

The polynucleotide of this invention was discovered in a cDNA libraryderived from human epithelioid sarcoma. It is structurally related tothe rat F-spondin family. It contains an open reading frame encoding aprotein of 331 amino acid residues. The protein exhibits the highestdegree of homology to rat F-spondin with 33.1% identity and 52.9%similarity over the entire amino acid stretch. The gene of the presentinvention shows the greatest homology at the nucleotide level to the ratF-spondin gene with 66% similarity and 66% identity. It is alsoimportant that the polypeptide of the present invention contains theconserved motif, WSXW, which is a potential binding sequence forpolypeptides in this family.

Northern blot analysis of the protein of the present invention showed abroad band at 1.6-1.9 kb in liver and lower level expression in kidney,lung, heart and placenta. Brain expression was barely detectable. Twolibraries which were constructed from tissues induced to undergoapoptosis, apoptotic t-cells (HTG) and TNF induced amniotic cells (HAU),had one clone in each. By extrapolation, NAF-1 was represented at least50 times more frequently in apoptotic t-cells expressed sequence tagsthan all normal and activated t-cell libraries. In the TNF inducedamniotic cells library, NAF-1 was detected 1 out of 2,414 expressedsequence tags versus 0 out of 3,595 expressed sequence tags for thenon-TNF treated amniotic cell library.

The NAF-1 cDNA contains an open reading frame encoding a polypeptide of35.8 kD. Amino acids 1-23 and 1-26 encode putative signal peptides.Accordingly, there are two species of predicted mature NAF-1polypeptides one having 311 and the other 314 amino acids. NAF-1 alsocontains a putative N-linked glycosylation site at position 303. Thehomology of NAF-1 to FSP covers amino acids 199-495 of the latterprotein. Thus, NAF-1 does not appear to be the human counterpart of therat FSP. NAF-1 contains only one TSR which begins at amino acid 278.This region is much more homologous to FSP type 1 repeats than to thoseof TSP, 38% versus 20%, respectively. The homology between the NAF-1 TSRand the six FSP type-1 repeats is shown in FIG. 3. The amino terminal277 amino acid of NAF-1 share homology to FSP but show no resemblance toany other known proteins.

In accordance with another aspect of the present invention there areprovided isolated polynucleotides encoding a mature polypeptideexpressed by the human cDNA contained in ATCC Deposit No. 97343,deposited with the American Type Culture Collection, 12301 Park LawnDrive; Rockville, Md. 20852, USA, on Nov. 20, 1995. The depositedmaterial is a pBluescript SK (−) (Stratagene, La. Jolla, Calif.) plasmidthat contains the full-length NAF-1 cDNA. The NAF-1 cDNA has been clonedinto the EcoRI, XhoI site.

The deposit has been made under the terms of the Budapest Treaty on theInternational Recognition of the Deposit of Micro-organisms for purposesof Patent Procedure. The strain will be irrevocably and withoutrestriction or condition released to the public upon the issuance of apatent. The deposit is provided merely as convenience to those of skillin the art and are not an admission that a deposit is required under 35U.S.C. §112. The sequence of the polynucleotide contained in thedeposited material, as well as the amino acid sequence of thepolypeptides encoded thereby, are controlling in the event of anyconflict with any description of sequences herein. A license may berequired to make, use or sell the deposited material, and no suchlicense is hereby granted.

The polynucleotide of the present invention may be in the form of RNA orin the form of DNA, which DNA includes cDNA, genomic DNA, and syntheticDNA. The DNA may be double-stranded or single-stranded, and if singlestranded may be the coding strand or non-coding (anti-sense) strand. Thecoding sequence which encodes the mature polypeptide may be identical tothe coding sequence shown in FIG. 1 (SEQ ID NO:1) or may be a differentcoding sequence which coding sequence, as a result of the redundancy ordegeneracy of the genetic code, encodes the same mature polypeptide asthe DNA of FIG. 1 (SEQ ID NO:1).

The polynucleotide which encodes for the mature polypeptide of FIG. 1(SEQ ID NO:2) may include, but is not limited to: only the codingsequence for the mature polypeptide; the coding sequence for the maturepolypeptide and additional coding sequence such as a leader or secretorysequence or a proprotein sequence; the coding sequence for the maturepolypeptide (and optionally additional coding sequence) and non-codingsequence, such as introns or non-coding sequence 5′ and/or 3′ of thecoding sequence for the mature polypeptide.

Thus, the term “polynucleotide encoding a polypeptide” encompasses apolynucleotide which includes only coding sequence for the polypeptideas well as a polynucleotide which includes additional coding and/ornon-coding sequence.

The present invention further relates to variants of the hereinabovedescribed polynucleotides which encode for fragments, analogs andderivatives of the polypeptide having the deduced amino acid sequence ofFIG. 1 (SEQ ID NO:2). The variant of the polynucleotide may be anaturally occurring allelic variant of the polynucleotide or anon-naturally occurring variant of the polynucleotide.

Thus, the present invention includes polynucleotides encoding the samemature polypeptide as shown in FIG. 1 (SEQ ID NO:2) as well as variantsof such polynucleotides which variants encode for a fragment, derivativeor analog of the polypeptide of FIG. 1 (SEQ ID NO:2). Such nucleotidevariants include deletion variants, substitution variants and additionor insertion variants.

As hereinabove indicated, the polynucleotide may have a coding sequencewhich is a naturally occurring allelic variant of the coding sequenceshown in FIG. 1 (SEQ ID NO:1). As known in the art, an allelic variantis an alternate form of a polynucleotide sequence which may have asubstitution, deletion or addition of one or more nucleotides, whichdoes not substantially alter the function of the encoded polypeptide.

The present invention also includes polynucleotides, wherein the codingsequence for the mature polypeptide may be fused in the same readingframe to a polynucleotide sequence which aids in expression andsecretion of a polypeptide from a host cell, for example, a leadersequence which functions as a secretory sequence for controllingtransport of a polypeptide from the cell. The polypeptide having aleader sequence is a preprotein and may have the leader sequence cleavedby the host cell to form the mature form of the polypeptide. Thepolynucleotides may also encode for a proprotein which is the matureprotein plus additional 5′ amino acid residues. A mature protein havinga prosequence is a proprotein and is an inactive form of the protein.Once the prosequence is cleaved an active mature protein remains. Thus,for example, the polynucleotide of the present invention may encode fora mature protein, or for a protein having a prosequence or for a proteinhaving both a prosequence and a presequence (leader sequence).

The polynucleotides of the present invention may also have the codingsequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence may be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptide fused to the markerin the case of a bacterial host, or, for example, the marker sequencemay be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

Most highly preferred are nucleic acid molecules encoding the matureprotein having the amino acid sequence shown in SEQ ID NO:2 as residues24-331 or 27-331, or the mature NAF-1 amino acid sequence encoded by thedeposited cDNA clone.

Also highly preferred are nucleic acid molecules encoding the TSR domainof the protein having the amino acid sequence shown in SEQ ID NO:8 orthe TSR domain of the NAF-1 amino acid sequence encoded by the depositedcDNA clone.

Thus, one aspect of the invention provides an isolated nucleic acidmolecule comprising a polynucleotide having a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequenceencoding a full-length NAF-1 polypeptide having the complete amino acidsequence in SEQ ID NO:2, or the complete amino acid sequence encoded bythe cDNA clone contained in the ATCC Deposit No. 97343; (b) a nucleotidesequence encoding a full-length NAF-1 polypeptide having the completeamino acid sequence in SEQ ID NO:2 excepting the N-terminal methionine(i.e., positions 2 to 331 of SEQ ID NO:2) or the complete amino acidsequence excepting the N-terminal methionine encoded by the cDNA clonecontained in the ATCC Deposit No. 97343; (c) a nucleotide sequenceencoding a predicted mature form of the NAF-1 polypeptide having theamino acid sequence at positions 24-331 or 27-331 in SEQ ID NO:2 or asencoded by the cDNA clone contained in the ATCC Deposit No. 97343; (d) anucleotide sequence encoding a polypeptide comprising the predicted TSRdomain of the NAF-1 polypeptide having the amino acid sequence atpositions 284-330 in SEQ ID NO:2 or as encoded by the cDNA clonecontained in the ATCC Deposit No. 97343; and (e) a nucleotide sequencecomplementary to any of the nucleotide sequences in (a), (b), (c) or (d)above.

Further embodiments of the invention include isolated nucleic acidmolecules that comprise a polynucleotide having a nucleotide sequence atleast 90% identical, and more preferably at least 95%, 96%, 97%, 98% or99% identical, to any of the nucleotide sequences in (a), (b), (c), (d)or (e), above, or a polynucleotide which hybridizes under stringenthybridization conditions to a polynucleotide in (a), (b), (c), (d) or(e), above. This polynucleotide which hybridizes does not hybridizeunder stringent hybridization conditions to a polynucleotide having anucleotide sequence consisting of only A residues or of only T residues.An additional nucleic acid embodiment of the invention relates to anisolated nucleic acid molecule comprising a polynucleotide which encodesthe amino acid sequence of an epitope-bearing portion of a NAF-1polypeptide having an amino acid sequence in (a), (b), (c), (d) or (e),above.

The present invention also relates to recombinant vectors, which includethe isolated nucleic acid molecules of the present invention, and tohost cells containing the recombinant vectors, as well as to methods ofmaking such vectors and host cells and for using them for production ofNAF-1 polypeptides or peptides by recombinant techniques.

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence encoding a NAF-1polypeptide is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding the NAF-1polypeptide. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. These mutations of thereference sequence may occur at the 5′ or 3′ terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence.

As a practical matter, whether any particular nucleic acid molecule isat least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, thenucleotide sequence shown in FIG. 1 or to the nucleotides sequence ofthe deposited cDNA clone can be determined conventionally using knowncomputer programs such as the Bestfit program (Wisconsin SequenceAnalysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711).Bestfit uses the local homology algorithm of Smith and Waterman,Advances in Applied Mathematics 2:482-489 (1981), to find the bestsegment of homology between two sequences. When using Bestfit or anyother sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set, of course,such that the percentage of identity is calculated over the full lengthof the reference nucleotide sequence and that gaps in homology of up to5% of the total number of nucleotides in the reference sequence areallowed.

The present application is directed to nucleic acid molecules at least90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequenceshown in FIG. 1 (SEQ ID NO:1) or to the nucleic acid sequence of thedeposited cDNA, irrespective of whether they encode a polypeptide havingNAF-1 activity. This is because even where a particular nucleic acidmolecule does not encode a polypeptide having NAF-1 activity, one ofskill in the art would still know how to use the nucleic acid molecule,for instance, as a hybridization probe or a polymerase chain reaction(PCR) primer. Uses of the nucleic acid molecules of the presentinvention that do not encode a polypeptide having NAF-1 activityinclude, inter alia, (1) isolating the NAF-1 gene or allelic variantsthereof in a cDNA library; (2) in situ hybridization (e.g., “FISH”) tometaphase chromosomal spreads to provide precise chromosomal location ofthe NAF-1 gene, as described in Verma et al., Human Chromosomes: AManual of Basic Techniques, Pergamon Press, New York (1988); andNorthern Blot analysis for detecting NAF-1 mRNA expression in specifictissues.

Preferred, however, are nucleic acid molecules having sequences at least90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequenceshown in FIG. 1 (SEQ ID NO:1) or to the nucleic acid sequence of thedeposited cDNA which do, in fact, encode a polypeptide having NAF-1protein activity. By “a polypeptide having NAF-1 activity” is intendedpolypeptides exhibiting activity similar, but not necessarily identical,to an activity of the mature protein of the invention, as measured in aparticular biological assay. For example, the NAF-1 protein of thepresent invention causes axonal neurite extension and promotes neuralcell adhesion. Such activity can be assayed as described in Klar, etal., Cell 69:95-110, incorporated herein by reference.

NAF-1 protein modulates axonal neurite extension and neural celladhesion in a dose-dependent manner in the above-described assay. Thus,“a polypeptide having NAF-1 protein activity” includes polypeptides thatalso exhibit any of the same neurite extension and neural cell adhesionpromoting activities in the above-described assays in a dose-dependentmanner. Although the degree of dose-dependent activity need not beidentical to that of the NAF-1 protein, preferably, “a polypeptidehaving NAF-1 protein activity” will exhibit substantially similardose-dependence in a given activity as compared to the NAF-1protein(i.e., the candidate polypeptide will exhibit greater activity or notmore than about 25-fold less and, preferably, not more than abouttenfold less activity relative to the reference NAF-1 protein).

Of course, due to the degeneracy of the genetic code, one of ordinaryskill in the art will immediately recognize that a large number of thenucleic acid molecules having a sequence at least 90%, 95%, 96%, 97%,98%, or 99% identical to the nucleic acid sequence of the deposited cDNAor the nucleic acid sequence shown in FIG. 1 (SEQ ID NO:1) will encode apolypeptide “having NAF-1 protein activity.” In fact, since degeneratevariants of these nucleotide sequences all encode the same polypeptide,this will be clear to the skilled artisan even without performing theabove described comparison assay. It will be further recognized in theart that, for such nucleic acid molecules that are not degeneratevariants, a reasonable number will also encode a polypeptide havingNAF-1 protein activity. This is because the skilled artisan is fullyaware of amino acid substitutions that are either less likely or notlikely to significantly effect protein function (e.g., replacing onealiphatic amino acid with a second aliphatic amino acid), as furtherdescribed below.

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

The present invention is further directed to nucleic acid moleculesencoding portions of the nucleotide sequences described herein as wellas to fragments of the isolated nucleic acid molecules described herein.In particular, the invention provides a polynucleotide having anucleotide sequence representing the portion of SEQ ID NO:1 whichconsists of positions 1-1010 of SEQ ID NO:1.

In addition, the invention provides nucleic acid molecules havingnucleotide sequences related to extensive portions of SEQ ID NO:1 whichhave been determined from the following related cDNA clones: HLHCE24R(shown as SEQ ID NO:16); HLHDR83R (shown as SEQ ID NO:17) and HPTSB36R(shown as SEQ ID NO:18).

Further, the invention includes a polynucleotide comprising any portionof at least about 30 nucleotides, preferably at least about 50nucleotides, of SEQ ID NO:1 from residue 1-650.

More generally, by a fragment of an isolated nucleic acid moleculehaving the nucleotide sequence of the deposited cDNA or the nucleotidesequence shown in FIG. 1 (SEQ ID NO:1) is intended fragments at leastabout 15 nt, and more preferably at least about 20 nt, still morepreferably at least about 30 nt, and even more preferably, at leastabout 40 nt in length which are useful as diagnostic probes and primersas discussed herein. Of course, larger fragments 50-300 nt in length arealso useful according to the present invention as are fragmentscorresponding to most, if not all, of the nucleotide sequence of thedeposited cDNA or as shown in FIG. 1 (SEQ ID NO:1). By a fragment atleast 20 nt in length, for example, is intended fragments which include20 or more contiguous bases from the nucleotide sequence of thedeposited cDNA or the nucleotide sequence as shown in FIG. 1 (SEQ IDNO:1). Preferred nucleic acid fragments of the present invention includenucleic acid molecules encoding epitope-bearing portions of the NAF-1polypeptide as identified in FIG. 4 and described in more detail below.

Fragments of the full length gene of the present invention may be usedas a hybridization probe for a cDNA library to isolate the full lengthcDNA and to isolate other cDNAs which have a high sequence similarity tothe gene or similar biological activity. Probes of this type preferablyhave at least 30 bases and may contain, for example, at least 50 bases.The probe may also be used to identify a cDNA clone corresponding to afull length transcript and a genomic clone or clones that contain thecomplete gene including regulatory and promotor regions, exons, andintrons. An example of a screen comprises isolating the coding region ofthe gene by using the known DNA sequence to synthesize anoligonucleotide probe. Labeled oligonucleotides having a sequencecomplementary to that of the gene of the present invention are used toscreen a library of human cDNA, genomic DNA or mRNA to determine whichmembers of the library the probe hybridizes to.

The present invention further relates to polynucleotides which hybridizeto the hereinabove-described sequences if there is at least 70%,preferably at least 90%, and more preferably at least 95%, 96%, 97%, 98%or 99% identity between the sequences. The present inventionparticularly relates to polynucleotides which hybridize under stringentconditions to the hereinabove-described polynucleotides. As herein used,the term “stringent hybridization conditions” is intended overnightincubation at 42° C. in a solution comprising: 50% formamide, 5×SSC (750mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6),5×Denhardt's solution, 10% dextran sulfate, and 20 μm/ml denatured,sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC atabout 65° C. The polynucleotides which hybridize to the hereinabovedescribed polynucleotides in a preferred embodiment encode polypeptideswhich either retain substantially the same biological function oractivity as the mature polypeptide encoded by the cDNAs of FIG. 1 (SEQID NO:1).

Alternatively, the polynucleotide may have at least 15 bases, preferablyat least 30 bases, and more preferably at least 50 bases which hybridizeto a polynucleotide of the present invention and which has an identitythereto, as described, and which may or may not retain activity. Forexample, such polynucleotides may be employed as probes for thepolynucleotide of SEQ ID NO:1, for example, for recovery of thepolynucleotide or as a diagnostic probe or as a PCR primer.

Thus, the present invention is directed to polynucleotides having atleast a 70% identity, preferably at least 90% and more preferably atleast a 95%, 96%, 97%, 98%, or 99% identity to a polynucleotide whichencodes the polypeptide of SEQ ID NO:2 and polynucleotides complementarythereto as well as portions thereof, which portions have at least 30consecutive bases and preferably at least 50 consecutive bases and topolypeptides encoded by such polynucleotides.

The present invention further relates to a polypeptide which has thededuced amino acid sequence of FIG. 1 (SEQ ID NO:2), as well asfragments, analogs and derivatives of such polypeptide.

To improve or alter the characteristics of NAF-1 polypeptides, proteinengineering may be employed. Recombinant DNA technology known to thoseskilled in the art can be used to create novel mutant proteins or“muteins including single or multiple amino acid substitutions,deletions, additions or fusion proteins. Such modified polypeptides canshow, e.g., enhanced activity or increased stability. In addition, theymay be purified in higher yields and show better solubility than thecorresponding natural polypeptide, at least under certain purificationand storage conditions.

For instance, for many proteins, including the extracellular domain of amembrane associated protein or the mature form(s) of a secreted protein,it is known in the art that one or more amino acids may be deleted fromthe N-terminus or C-terminus without substantial loss of biologicalfunction. For instance, Ron et al., J. Biol. Chem., 268:2984-2988 (1993)reported modified KGF proteins that had heparin binding activity even if3, 8, or 27 amino-terminal amino acid residues were missing. In thepresent case, since the protein of the invention contains a TSR repeat,deletions of N-terminal amino acids up to the cysteine at position 284(C284) of SEQ ID NO:2 may retain some biological activity such as theability to promote cell adhesion, however, additional deletionsincluding C284 would not be expected to retain such biologicalactivities because it is known that this residue in the TSR repeat isrequired for secondary structure necessary to promote cell adhesion.

However, even if deletion of one or more amino acids from the N-terminusof a protein results in modification or loss of one or more biologicalfunctions of the protein, other biological activities may still beretained. Thus, the ability of the shortened protein to induce and/orbind to antibodies which recognize the complete or TSR domain of theprotein generally will be retained when less than the majority of theresidues of the complete or TSR domain are removed from the N-terminus.Whether a particular polypeptide lacking N-terminal residues of acomplete protein retains such immunologic activities can readily bedetermined by routine methods described herein and otherwise known inthe art.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the amino terminus of the amino acidsequence of the NAF-1 shown in SEQ ID NO:2, up to the C284 residue, andpolynucleotides encoding such polypeptides. In particular, the presentinvention provides polypeptides comprising the amino.

Similarly, many examples of biologically functional C-terminal deletionmuteins are known. For instance, interferon gamma shows up to ten timeshigher activities by deleting 8-10 amino acid residues from the carboxyterminus of the protein (Döbeli et al., J. Biotechnology 7:199-216(1988). However, even if deletion of one or more amino acids from theC-terminus of a protein results in modification of loss of one or morebiological ftunctions of the protein, other biological activities maystill be retained. Thus, the ability of the shortened protein to induceand/or bind to antibodies which recognize the complete or TSR domain ofthe protein generally will be retained when less than the majority ofthe residues of the complete or TSR domain protein are removed from theC-terminus. Whether a particular polypeptide lacking C-terminal residuesof a complete protein retains such immunologic activities can readily bedetermined by routine methods described herein and otherwise known inthe art.

Accordingly, the present invention further provides polypeptides havingone or more residues from the carboxy terminus of the amino acidsequence of the NAF-1 shown in SEQ ID NO:2, up to the C330 residue ofSEQ ID NO:2, and polynucleotides encoding such polypeptides. Inparticular, the present invention provides polypeptides having the aminoacid sequence of residues 1-m of the amino acid sequence in SEQ ID NO:2,where m is either 330 or 331, and C330 is the position of the firstresidue from the C-terminus of the complete NAF-1 polypeptide (shown inSEQ ID NO:2) believed to be required for cell adhesion of the NAF-1protein.

The invention also provides polypeptides having one or more amino acidsdeleted from both the amino and the carboxyl termini, which may bedescribed generally as having residues n-m of SEQ ID NO:2, where n and mare integers as described above.

Also included are a nucleotide sequence encoding a polypeptideconsisting of a portion of the complete NAF-1 amino acid sequenceencoded by the cDNA clone contained in ATCC Deposit No. 97343, wherethis portion excludes from 1 to about 283 amino acids from the aminoterminus of the complete amino acid sequence encoded by the cDNA clonecontained in ATCC Deposit No. 97343, or 1 amino acid from the carboxyterminus, or any combination of the above amino terminal and carboxyterminal deletions, of the complete amino acid sequence encoded by thecDNA clone contained in ATCC Deposit No. 97343. Polynucleotides encodingall of the above deletion mutant polypeptide forms also are provided.

In addition to terminal deletion forms of the protein discussed above,it also will be recognized by one of ordinary skill in the art that someamino acid sequences of the NAF-1 polypeptide can be varied withoutsignificant effect of the structure or function of the protein. If suchdifferences in sequence are contemplated, it should be remembered thatthere will be critical areas on the protein which determine activity.

Thus, the invention further includes variations of the NAF-1 polypeptidewhich show substantial NAF-1 polypeptide activity or which includeregions of NAF-1 protein such as the protein portions discussed below.Such mutants include deletions, insertions, inversions, repeats, andtype substitutions selected according to general rules known in the artso as have little effect on activity. For example, guidance concerninghow to make phenotypically silent amino acid substitutions is providedin Bowie, J. U. et al., “Deciphering the Message in Protein Sequences:Tolerance to Amino Acid Substitutions,” Science 247:1306-1310 (1990),wherein the authors indicate that there are two main approaches forstudying the tolerance of an amino acid sequence to change. The firstmethod relies on the process of evolution, in which mutations are eitheraccepted or rejected by natural selection. The second approach usesgenetic engineering to introduce amino acid changes at specificpositions of a cloned gene and selections or screens to identifysequences that maintain functionality.

As the authors state, these studies have revealed that proteins aresurprisingly tolerant of amino acid substitutions. The authors furtherindicate which amino acid changes are likely to be permissive at acertain position of the protein. For example, most buried amino acidresidues require nonpolar side chains, whereas few features of surfaceside chains are generally conserved. Other such phenotypically silentsubstitutions are described in Bowie, J. U. et al., supra, and thereferences cited therein. Typically seen as conservative substitutionsare the replacements, one for another, among the aliphatic amino acidsAla, Val, Leu and Ile; interchange of the hydroxyl residues Ser and Thr,exchange of the acidic residues Asp and Glu, substitution between theamide residues Asn and Gln, exchange of the basic residues Lys and Argand replacements among the aromatic residues Phe, Tyr.

Thus, the fragment, derivative or analog of the polypeptide of SEQ IDNO:2, or that encoded by the deposited cDNA, may be (i) one in which oneor more of the amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code, or (ii) one in which one or more of theamino acid residues includes a substituent group, or (iii) one in whichthe mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the above form of the polypeptide, such as an IgG Fc fusionregion peptide or leader or secretory sequence or a sequence which isemployed for purification of the above form of the polypeptide or aproprotein sequence. Such fragments, derivatives and analogs are deemedto be within the scope of those skilled in the art from the teachingsherein Thus, the NAF-1 of the present invention may include one or moreamino acid substitutions, deletions or additions, either from naturalmutations or human manipulation. As indicated, changes are preferably ofa minor nature, such as conservative amino acid substitutions that donot significantly affect the folding or activity of the protein (seeTable 1).

TABLE 1 Conservative Amino Acid Substitutions. Aromatic PhenylalanineTryptophan Tyrosine Hydrophobic Leucine Isoleucine Valine PolarGlutamine Asparagine Basic Arginine Lysine Histidine Acidic AsparticAcid Glutamic Acid Small Alanine Serine Threonine Methionine Glycine

Amino acids in the NAF-1 protein of the present invention that areessential for function can be identified by methods known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244:1081-1085 (1989)). The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as receptor binding or in vitro or in vitro proliferativeactivity.

Of special interest are substitutions of charged amino acids with othercharged or neutral amino acids which may produce proteins with highlydesirable improved characteristics, such as less aggregation.Aggregation may not only reduce activity but also be problematic whenpreparing pharmaceutical formulations, because aggregates can beimmunogenic (Pinckard et al., Clin. Exp. Immunol 2:331-340 (1967);Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev.Therapeutic Drug Carrier Systems 10:307.

FIG. 3 shows the consensus TSR sequence (Sequence ID NO:14). Preferredmutants having increased cell adhesion activity are those withsubstitutions making the NAF-1 polypeptides more similar to theconsensus sequence.

The terms “fragment,” “derivative” and “analog” when referring to thepolypeptide of FIG. 1 (SEQ ID NO:2), means a polypeptide which retainsessentially the same biological function or activity as suchpolypeptide. Thus, an analog includes a proprotein which can beactivated by cleavage of the proprotein portion to produce an activemature polypeptide.

The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide,preferably a recombinant polypeptide.

The fragment, derivative or analog of the polypeptide of FIG. 1 (SEQ IDNO:2) may be (i) one in which one or more of the amino acid residues aresubstituted with a conserved or non-conserved amino acid residue(preferably a conserved amino acid residue) and such substituted aminoacid residue may or may not be one encoded by the genetic code, or (ii)one in which one or more of the amino acid residues includes asubstituent group, or (iii) one in which the mature polypeptide is fusedwith another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol), or (iv) one in whichthe additional amino acids are fused to the mature polypeptide, such asa leader or secretory sequence or a sequence which is employed forpurification of the mature polypeptide or a proprotein sequence. Suchfragments, derivatives and analogs are deemed to be within the scope ofthose skilled in the art from the teachings herein.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The invention further provides an isolated NAF-1 polypeptide comprisingan amino acid sequence selected from the group consisting of: (a) theamino acid sequence of the full-length NAF-1 polypeptide having thecomplete amino acid sequence shown in SEQ ID NO:2 or the complete aminoacid sequence excepting the N-terminal methionine encoded by the cDNAclone contained in the ATCC Deposit No. 97343; (b) the amino acidsequence of the full-length NAF-1 polypeptide having the complete aminoacid sequence shown in SEQ ID NO:2 excepting the N-terminal methionine(i.e., positions 1-331 of SEQ ID NO:2) or the complete amino acidsequence excepting the N-terminal methionine encoded by the cDNA clonecontained in the ATCC Deposit No. 97343; (c) the amino acid sequence ofthe mature NAF-1 polypeptide having the amino acid sequence of residues24-331 or 27-331 in SEQ ID NO:2, or the mature NAF-1 amino acid sequenceas encoded by the cDNA clone contained in ATCC Deposit No. 97343; and(d) the amino acid sequence of the TSR domain of NAF-1 having the aminoacid sequence of residues 284 to 330 of SEQ ID NO:2, or the amino acidsequence of the TSR domain of NAF-1 encoded by the cDNA clone containedin ATCC Deposit No. 97343.

Further polypeptides of the present invention include polypeptides whichhave at least 90% similarity, more preferably at least 95% similarity,and still more preferably at least 96%, 97%, 98% or 99% similarity tothose described above. The polypeptides of the invention also comprisethose which are at least 80% identical, more preferably at least 90% or95% identical, still more preferably at least 96%, 97%, 98% or 99%identical to the polypeptide encoded by the deposited cDNA or to thepolypeptide of SEQ ID NO:2, and also include portions of suchpolypeptides with at least 30 amino acids and more preferably at least50 amino acids.

By “% similarity” for two polypeptides is intended a similarity scoreproduced by comparing the amino acid sequences of the two polypeptidesusing the Bestfit program (Wisconsin Sequence Analysis Package, Version8 for Unix, Genetics Computer Group, University Research Park, 575Science Drive, Madison, Wis. 53711) and the default settings fordetermining similarity. Bestfit uses the local homology algorithm ofSmith and Waterman (Advances in Applied Mathematics 2:482-489, 1981) tofind the best segment of similarity between two sequences.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a reference amino acid sequence of a NAF-1polypeptide is intended that the amino acid sequence of the polypeptideis identical to the reference sequence except that the polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the reference amino acid of the NAF-1 polypeptide. Inother words, to obtain a polypeptide having an amino acid sequence atleast 95% identical to a reference amino acid sequence, up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to 5%of the total amino acid residues in the reference sequence may beinserted into the reference sequence. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

As a practical matter, whether any particular polypeptide is at least90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the aminoacid sequence shown in SEQ ID NO:2 or to the amino acid sequence encodedby deposited cDNA clone an be determined conventionally using knowncomputer programs such the Bestfit program (Wisconsin Sequence AnalysisPackage, Version 8 for Unix, Genetics Computer Group, UniversityResearch Park, 575 Science Drive, Madison, Wis. 53711). When usingBestfit or any other sequence alignment program to determine whether aparticular sequence is, for instance, 95% identical to a referencesequence according to the present invention, the parameters are set, ofcourse, such that the percentage of identity is calculated over the fulllength of the reference amino acid sequence and that gaps in homology ofup to 5% of the total number of amino acid residues in the referencesequence are allowed.

In another aspect, the invention provides a peptide or polypeptidecomprising an epitope-bearing portion of a polypeptide of the invention.The epitope of this polypeptide portion is an immunogenic or antigenicepitope of a polypeptide of the invention. An “immunogenic epitope” isdefined as a part of a protein that elicits an antibody response whenthe whole protcon is the immunogen. On the other hand, a region of aprotein molecule to which an antibody can bind is defined as an“antigenic epitope.” The number of immunogenic epitopes of a proteingenerally is less than the number of antigenic epitopes. See, forinstance, Geysen et aL, Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983).

As to the selection of peptides or polypeptides bearing an antigenicepitope (i.e., that contain a region of a protein molecule to which anantibody can bind), it is well known in that art that relatively shortsynthetic peptides that mimic part of a protein sequence are routinelycapable of eliciting an antiserum that reacts with the partiallymimicked protein. See, for instance, Sutcliffe, J. G., Shinnick, T. M.,Green, N. and Learner, R. A. (1983) “Antibodies that react withpredetermined sites on proteins,” Science, 219:660-666. Peptides capableof eliciting protein-reactive sera are frequently represented in theprimary sequence of a protein, can be characterized by a set of simplechemical rules, and are confined neither to immunodominant regions ofintact proteins (i.e., immunogenic epitopes) nor to the amino orcarboxyl terminals. Antigenic epitope-bearing peptides and polypeptidesof the invention are therefore useful to raise antibodies, includingmonoclonal antibodies, that bind specifically to a polypeptide of theinvention. See, for instance, Wilson et al., Cell 37:767-778 (1984) at777.

Antigenic epitope-bearing peptides and polypeptides of the inventionpreferably contain a sequence of at least seven, more preferably atleast nine and most preferably between about 15 to about 30 amino acidscontained within the amino acid sequence of a polypeptide of theinvention. Non-limiting examples of antigenic polypeptides or peptidesthat can be used to generate NAF-1-specific antibodies include: apolypeptide comprising amino acid residues from about Ak-75 to aboutArg-100); a polypeptide comprising amino acid residues from aboutLev-168 to about Ala-180); a polypeptide comprising amino acid residuesfrom about Thr-204 to about Tyr 226); and a polypeptide comprising aminoacid residues from about Lev-258 to about Ser-281); and a polypeptidecomprising amino acid residues from about Gly-291 to about Pro-327).These polypeptide fragments have been determined to bear antigenicepitopes of the NAF-1 protein by the analysis of the Jameson-Wolfantigenic index, as shown in FIG. 4, above.

The epitope-bearing peptides and polypeptides of the invention may beproduced by any conventional means. See, e.g., Houghten, R. A. (1985)“General method for the rapid solid-phase synthesis of large numbers ofpeptides: specificity of antigen-antibody interaction at the level ofindividual amino acids.” Proc. Natl. Acad. Sci. USA 82:5131-5135; this“Simultaneous Multiple Peptide Synthesis (SMPS)” process is furtherdescribed in U.S. Pat. No. 4,631,211 to Houghten et aL (1986).

Epitope-bearing peptides and polypeptides of the invention are used toinduce antibodies according to methods well known in the art. See, forinstance, Sutcliffe et al., supra; Wilson et al., supra; Chow, M. etal., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle, F. J. et al., J.Gen. Virol 66:2347-2354 (1985). Immunogenic epitope-bearing peptides ofthe invention, i.e., those parts of a protein that elicit an antibodyresponse when the whole protein is the imnmunogen, are identifiedaccording to methods known in the art. See, for instance, Geysen et al.,supra. Further still, U.S. Pat. No. 5,194,392 to Geysen (1990) describesa general method of detecting or determining the sequence of monomers(amino acids or other compounds) which is a topological equivalent ofthe epitope (i.e., a “mimotope”) which is complementary to a particularparatope (antigen binding site) of an antibody of interest. Moregenerally, U.S. Pat. No. 4,433,092 to Geysen (1989) describes a methodof detecting or determining a sequence of monomers which is atopographical equivalent of a ligand which is complementary to theligand binding site of a particular receptor of interest. Similarly,U.S. Pat. No. 5,480,971 to Houghten, R. A. et al. (1996) on PeralkylatedOligopeptide Mixtures discloses linear C1-C7-alkyl peralkylatedoligopeptides and sets and libraries of such peptides, as well asmethods for using such oligopeptide sets and libraries for determiningthe sequence of a peralkylated oligopeptide that preferentially binds toan acceptor molecule of interest. Thus, non-peptide analogs of theepitope-bearing peptides of the invention also can be made routinely bythese methods.

Fragments or portions of the polypeptides of the present invention maybe employed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, the fragments may be employed asintermediates for producing the full-length polypeptides. Fragments orportions of the polynucleotides of the present invention may be used tosynthesize full-length polynucleotides of the present invention.

The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed ortransfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the genes of the present invention. Theculture conditions, such as temperature, pH and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct mRNAsynthesis. As representative examples of such promoters, there may bementioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or moreselectable marker genes to provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E coli.

The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein.

As representative examples of appropriate hosts, there may be mentioned:bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium;fungal cells, such as yeast; insect cells such as Drosophila S2 andSpodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma;adenoviruses; plant cells, etc. The selection of an appropriate host isdeemed to be within the scope of those skilled in the art from theteachings herein.

More particularly, the present invention also includes recombinantconstructs comprising one or more of the sequences as broadly describedabove. The constructs comprise a vector, such as a plasmid or viralvector, into which a sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example; Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pBS, pD10, phagescript, psiX174, pBluescript SK, pBSKS, pNH8A, pNH16a,pNH18A, pNH46A (Stratagene); pTRC99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia); Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene)pSVK3, pBPV, pMSG, pSVL (Pharmnacia). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

Promoter regions can be selected from any desired gene using CAT Fat(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lac, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cellscontaining the above-described constructs. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (Davis, L., Dibner, M., Battey, I.,Basic Methods in Molecular Biology, (1986)).

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), thedisclosure of which is hereby incorporated by reference.

Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples include the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

Useful expression vectors for bacterial use are constructed by insertinga structural DNA sequence encoding a desired protein together withsuitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

As a representative but nonlimiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis.,USA). These pBR322 “backbone” sections are combined with an appropriatepromoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, such methods arewell known to those skilled in the art.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman,Cell, 23:175 (1981), and other cell lines capable of expressing acompatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mamnmalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

The polypeptide can be recovered and purified from recombinant cellcultures by methods including ammonium sulfate or ethanol precipitation,acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Protein refolding steps can be used, as necessary, incompleting configuration of the mature protein. Finally, highperformance liquid chromatography (HPLC) can be employed for finalpurification steps.

The polypeptides of the present invention may be a naturally purifiedproduct, or a product of chemical synthetic procedures, or produced byrecombinant techniques front a prokaryotic or eukaryotic host (forexample, by bacterial, yeast, higher plant, insect and mammalian cellsin culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay also include an initial methionine amino acid residue.

NAF-1 may be employed to treat spinal cord injuries or damage toperipheral nerves by increasing spinal cord and sensory neuronattachment and neurite outgrowth.

NAF-1 may also be employed to inhibit tumor cell metastases induced bysmall cell carcinoma. The NAF-1 gene and gene product of the presentinvention may also be employed to reduce primary tumor growth,metastatic potential and angiogenesis in human breast carcinoma cells.

The NAF-1 gene and gene product of the present invention may also beemployed to promote wound healing due to its ability to promotecell-cell interaction and cell adhesion.

NAF-1 may also be employed to modulate hemostasis.

The polynucleotides and polypeptides of the present invention may beemployed as research reagents and materials for discovery of treatmentsand diagnostics to human disease.

This invention provides a method for identification of the receptor forNAF-1. The gene encoding methods known to those of skill in the art, forexample, ligand panning and FACS sorting (Coligan, et al., CurrentProtocols in Immun., 1(2), Chapter 5, (1991)). Preferably, expressioncloning is employed wherein polyadenylated RNA is prepared from a cellresponsive to NAF-1, and a cDNA library created from this RNA is dividedinto pools and used to transfect COS cells or other cells that are notresponsive to NAF-1. Transfected cells which are grown on glass slidesare exposed to labeled NAF-1 ligand. NAF-1 can be labeled by a varietyof means including iodination or inclusion of a recognition site for asite-specific protein kinase. Following fixation and incubation, the,slides are subjected to auto-radiographic analysis. Positive pools areidentified and sub-pools are prepared and re-transfected using aniterative sub-pooling and re-screening process, eventually yielding asingle clone that encodes the putative receptor. As an alternativeapproach for receptor identification, labeled ligand can bephotoaffinity linked with cell membrane or extract preparations thatexpress the receptor molecule. Cross-linked material is resolved by PAGEand exposed to X-ray film. The labeled complex containing theligand-receptor can be excised, resolved into peptide fragments, andsubjected to protein microsequencing. The amino acid sequence obtainedfrom microsequencing would be used to design a set of degenerateoligonucleotide probes to screen a cDNA library to identify the geneencoding the putative receptor.

This invention provides a method of screening compounds to identifythose which are agonists to or antagonists to NAF-1. The identificationof both type compounds would involve a neurite outgrowth assay. COScells (5×10⁸) are transfected with NAF-1/pcDNA-1 (Invitrogen, Inc.) andconditioned medium is collected. NAF^(myc) is affinity purified on amonoclonal anti-myc (9E10) affinity-purified F-spondin^(myc) (20 mg/ml)is absorbed onto nitrocellulose (Lenimon et al., 1989). For controls,parental COS cell-conditioned medium is purified on the same column andused as a substrate on nitrocellulose. The nitrocellulose is thenblocked with BSA (10 mg/ml), which provided a further control forbackground neurite outgrowth. rAT E14 DRG neurons are plated onimmobilized protein substrates at a density of 2-10×10⁴ cells per 35 mmtissue culture dish (Nunc) and grown for 14 hr. Cultures are then fixedin 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, andstained using MAb 3A10 (Furley et al., 1990; available fromDevelopmental Studies Hybridoma Bank), which recognizes a neuronalfilament-associated protein and serves as a marker for fine neurites.Neuronal cell bodies and neurites are visualized by indirectimmunofluorescence on a Zeiss Axioplan microscope. Neurite lengths aremeasured as the distance from the edge of the soma (sharply defined by3A10 fluorescence) to the tip of its longest neurite. Neurite lengthsare measured if the entire length of the neurite could be unambiguouslyidentified. About 25 neurites are measurable within each protein-coatedarea (3-4 mm²).

Rat e13 dorsal spinal cord neurons can also be assayed by plating thedissociated cells on immobilized protein substrate at a density of 10⁶cells per 35 mm tissue culture dish (Nunc). After 1 hr. the cultures arewashed twice with PBS and fixed in 4% paraformaldehyde. Cells arecounted on a Zeiss Axioplan microscope at 400×magnification. Tenindependent counts are taken from each experiment.

An alternative example of identifying agonists and antagonists to thepolypeptide of the present invention includes expressing the NAF-1receptor from a mammalian cell or membrane preparation and incubatingthat receptor with labeled NAF-1 in the presence of a compound. Theability of a compound to enhance or block the interaction is thenquantified. Alternatively, the response of a known second messengersystem following interaction of NAF-1 and its receptor would be measuredand compared in the presence or absence of the compound. Such secondmessenger systems include, but are not limited, cAMP guanylate cyclase,ion channels or phosphoinositide hydrolysis.

Potential antagonists include an antibody, or in some cases, anoligopeptide, which binds to the polypeptide. Alternatively, a potentialantagonist may be a closely related protein which binds to the receptorsites, however, they are inactive forms of the polypeptide and therebyprevent the action of NAF-1 since receptor sites are occupied.

Another potential antagonist is an antisense construct prepared usingantisense technology. Antisense technology can be used to control geneexpression through triple-helix formation or antisense DNA or RNA, bothof which methods are based on binding of a polynucleotide to DNA or RNA.For example, the 5′ coding portion of the polynucleotide sequence, whichencodes for the mature polypeptides of the present invention, is used todesign an antisense RNA oligonucleotide of from about 10 to 40 basepairs in length. A DNA oligonucleotide is designed to be complementaryto a region of the gene involved in transcription (triple helix—see Leeet al., Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science, 241:456(1988); and Dervan et al., Science, 251:1360 (1991)), thereby preventingtranscription and the production of NAF-1. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into NAF-1 polypeptide (Antisense—Okano, J.Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Theoligonucleotides described above can also be delivered to cells suchthat the antisense RNA or DNA may be expressed in vivo to inhibitproduction of NAF-1.

Potential antagonists include a small molecule which binds to andoccupies the catalytic site of the polypeptide thereby making thecatalytic site inaccessible to substrate such that normal biologicalactivity is prevented. Examples of small molecules include but are notlimited to small peptides or peptide-like molecules.

The antagonists may be employed to treat malarial infection induced byPlasinodium falciparum. During malarial infection, the polypeptide ofthe present invention may promote adhesion of parasitized red cells toendothelial cells and, therefore, antagonists would inhibit this actionand prevent malaria. The antagonists may also be employed to treatcancer, for example, in blocking metastasis by inhibiting cell adhesion.

The polypeptides of the present invention or antagonists and agonistsmay be employed in combination with a suitable pharmaceutical carrier.Such compositions comprise a therapeutically effective amount of thepolypeptide or antagonists or agonist, and a pharmaceutically acceptablecarrier or excipient. Such a carrier includes but is not limited tosaline, buffered saline, dextrose, water, glycerol, ethanol, andcombinations thereof. The formulation should suit the mode ofadministration.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, thepolypeptides of the present invention or agonists or antagonists may beemployed in conjunction with other therapeutic compounds.

The pharmaceutical compositions may be administered in a convenientmanner such as by the oral, topical, parenterally, intravenous,intraperitoneal, intramuscular, subcutaneous, intranasal or intradermalroutes. The pharmaceutical compositions are administered in an amountwhich is effective for treating and/or prophylaxis of the specificindication. In general, they are administered in an amount of at leastabout 10 μg/kg body weight and in most cases they will be administeredin an amount not in excess of about 8 mg/Kg body weight per day. In mostcases, the dosage is from about 10 μg/kg to about 1 mg/kg body weightdaily, taking into account the routes of admninistration, symptoms, etc.

The NAF-1 polypeptides and agonists and antagonists which arepolypeptides may also be employed in accordance with the presentinvention by expression of such polypeptides in vivo, which is oftenreferred to as “gene therapy.”

Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with theengineered cells then being provided to a patient to be treated with thepolypeptide. Such methods are well-known in the art and are apparentfrom the teachings herein. For example, cells may be engineered by theuse of a retroviral plasmid vector containing RNA encoding a polypeptideof the present invention.

Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by, for example, procedures known in the art. Forexample, a packaging cell is transduced with a retroviral plasmid vectorcontaining RNA encoding a polypeptide of the present invention such thatthe packaging cell now produces infectious viral particles containingthe gene of interest. These producer cells may be administered to apatient for engineering cells in vivo and expression of the polypeptidein vivo. These and other methods for administering a polypeptide of thepresent invention by such method should be apparent to those skilled inthe art from the teachings of the present invention.

Retroviruses from which the retroviral plasmid vectors hereinabovementioned may be derived include, but are not limited to, Moloney MurineLeukemia Virus, spleen necrosis virus, retroviruses such as Rous SarcomaVirus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemiavirus, human immunodeficiency virus, adenovirus, MyeloproliferativeSarcoma Virus, and mammary tumor virus. In one embodiment, theretroviral plasmid vector is derived from Moloney Murine Leukemia Virus.

The vector includes one or more promoters. Suitable promoters which maybe employed include, but are not limited to, the retroviral LTR; theSV40 promoter; and the human cytomegalovirus (CMV) promoter described inMiller, et al., Biotechniques, Vol. 7, No. 9, 980-990 (1989), or anyother promoter (e.g., cellular promoters such as eukaryotic cellularpromoters including, but not limited to, the histone, pot III, andb-actin promoters). Other viral promoters which may be employed include,but are not limited to, adenovirus promoters, thymidine kinase (TK)promoters, and B19 parvovirus promoters. The selection of a suitablepromoter will be apparent to those skilled in the art from the teachingscontained herein.

The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter. Suitablepromoters which may be employed include, but are not limited to,adenoviral promoters, such as the adenoviral major late promoter; orhetorologous promoters, such as the cytomegalovirus. (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter, the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRshereinabove described); the b-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe gene encoding the polypeptide.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, y-2,y-AM, PA12, T19-14X, VT-19-17-H2, yCRE, yCRIP, GP+E-86, GP+envAm12, andDAN cell lines as described in Miller, Human Gene Therapy, Vol. 1, pgs.5-14 (1990), which is incorporated herein by reference in its entirety.The vector may transduce the packaging cells through any means known inthe art. Such means include, but are not limited to, electroporation,the use of liposomes, and CaPO₄ precipitation. In one alternative, theretroviral plasmid vector may be encapsulated into a liposome, orcoupled to a lipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particleswhich include the nucleic acid sequence(s) encoding the polypeptides.Such retroviral vector particles then may be employed, to transduceeukaryotic cells, either in vitro or in vivo. The transduced eukaryoticcells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

This invention is also related to the use of the gene of the presentinvention as a diagnostic. Detection of a mutated form of the gene willallow a diagnosis of a disease or a susceptibility to a disease whichresults from underexpression of NAF-1, for example, tumor metastases andtumor angiogenesis.

Individuals carrying mutations in the gene of the present invention maybe detected at the DNA level by a variety of techniques. Nucleic acidsfor diagnosis may be obtained from a patient's cells, including but notlimited to blood, urine, saliva, tissue biopsy and autopsy material. Thegenomic DNA may be used directly for detection or may be amplifiedenzymatically by using PCR (Saiki et al., Nature, 324:163-166 (1986))prior to analysis. RNA or cDNA may also be used for the same purpose. Asan example, PCR primers complementary to the nucleic acid encoding NAF-1can be used to identify and analyze mutations. For example, deletionsand insertions can be detected by a change in size of the amplifiedproduct in comparison to the normal genotype. Point mutations can beidentified by hybridizing amplified DNA to radiolabeled RNA oralternatively, radiolabeled antisense DNA sequences. Perfectly matchedsequences can be distinguished from mismatched duplexes by RNase Adigestion or by differences in melting temperatures.

Sequence differences between the reference gene and genes havingmutations may be revealed by the direct DNA sequencing method. Inaddition, cloned DNA segments may be employed as probes to detectspecific DNA segments. The sensitivity of this method is greatlyenhanced when combined with PCR. For example, a sequencing primer isused with double-stranded PCR product or a single-stranded templatemolecule generated by a modified PCR. The sequence determination isperformed by conventional procedures with radiolabeled nucleotide or byautomatic sequencing procedures with fluorescent-tags.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments ingels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230:1242 (1985)).

Sequence changes at specific locations may also be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401 (1985)).

Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,Restriction Fragment Length Polymorphisms (RFLP)) and Southern blottingof genomic DNA.

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations can also be detected by in situ analysis.

The present invention also relates to a diagnostic assay for detectingaltered levels of the polypeptide of the present invention in varioustissues since an over-expression of the proteins compared to normalcontrol tissue samples can detect the presence of NAF-1 and conditionsrelated to an overexpression of NAF-1, for example, tumor metastases andangiogenesis. Assays used to detect levels of the polypeptide of thepresent invention in a sample derived from a host are well-known tothose of skill in the art and include radioimmunoassays,competitive-binding assays, Western Blot analysis and preferably anELISA assay. An ELISA assay initially comprises preparing an antibodyspecific to the NAF-1 antigen, preferably a monoclonal antibody. Inaddition a reporter antibody is prepared against the monoclonalantibody. To the reporter antibody is attached a detectable reagent suchas radioactivity, fluorescence or in this example a horseradishperoxidase enzyme. A sample is now removed from a host and incubated ona solid support, e.g. a polystyrene dish, that binds the proteins in thesample. Any free protein binding sites on the dish are then covered byincubating with a non-specific protein such as bovine serum albumin.Next, the monoclonal antibody is incubated in the dish during which timethe monoclonal antibodies attached to any of the polypeptide of thepresent invention attached to the polystyrene dish. All unboundmonoclonal antibody is washed out with buffer. The reporter antibodylinked to horseradish peroxidase is now placed in the dish resulting inbinding of the reporter antibody to any monoclonal antibody bound to thepolypeptide of the present invention. Unattached reporter antibody isthen washed out. Peroxidase substrates are then added to the dish andthe amount of color developed in a given time period is a measurement ofthe amount of the polypeptide of the present invention present in agiven volume of patient sample when compared against a standard curve.

A competition assay may be employed wherein antibodies specific to thepolypeptide of the present invention are attached to a solid support andlabeled NAF-1 and a sample derived from the host are passed over thesolid support and the amount of label detected attached to the solidsupport can be correlated to a quantity of the polypeptide of thepresent invention in the sample.

The sequences of the present invention are also valuable for chromosomeidentification. The sequence is specifically targeted to and canhybridize with a particular location on an individual human chromosome.Moreover, there is a current need for identifying particular sites onthe chromosome. Few chromosome marking reagents based on actual sequencedata (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp) from the cDNA. Computer analysis of the 3′untranslated region of the gene is used to rapidly select primers thatdo not span more than one exon in the genomic DNA, thus complicating theamplification process. These primers are then used for PCR screening ofsomatic cell hybrids containing individual human chromosomes. Only thosehybrids containing the human gene corresponding to the primer will yieldan amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of fragments from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that cansimilarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphasechromosomal spread can be used to provide a precise chromosomal locationin one step. This technique can be used with cDNA having at least 50 or60 bases. For a review of this technique, see Verma et al., HumanChromosomes: a Manual of Basic Techniques, Pergamon Press, New York(1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man (available on line through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddiseases that have been mapped to the sane chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

The polypeptides, their fragments or other derivatives, or analogsthereof, or cells expressing them can be used as an immunogen to produceantibodies thereto. These antibodies can be, for example, polyclonal ormonoclonal antibodies. The present invention also includes chimeric,single chain, and humanized antibodies, as well as Fab fragments, or theproduct of an Fab expression library. Various procedures known in theart may be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma Technique (Kohler and Milstein, 1975,Nature, 256:495-497), the trioma technique, the human B-cell hybridomatechnique (Kozbor et al., 1983, Immunology Today 4:72), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole, etal., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic polypeptide products of this invention. Also, transgenicmice may be used to express humanized antibodies to immunogenicpolypeptide products of this invention.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptide or to purify the polypeptide of thepresent invention by attachment of the antibody to a solid support forisolation and/or purification by affinity chromatography.

The present invention will be further described with reference to thefollowing examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

In order to facilitate understanding of the following examples certainfrequently occurring methods and/or terms will be described. “Plasmids”are designated by a lower case p preceded and/or followed by capitalletters and/or numbers. The starting plasmids herein are eithercommercially available, publicly available oh an unrestricted basis, orcan be constructed from available plasmids in accord with publishedprocedures. In addition, equivalent plasmids to those described areknown in the art and will be apparent to the ordinarily skilled artisan.

“Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 mg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 ml of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 mgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37∞ C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

Size separation of the cleaved fragments is performed using 8 percentpolyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res.,8:4057 (1980).

“Oligonucleotides” refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5′ phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

“Ligation” refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (Maniatis, T., et al., Id.,p. 146). Unless otherwise provided, ligation may be accomplished usingknown buffers and conditions with 10 units of T4 DNA ligase (“ligase”)per 0.5 mg of approximately equimolar amounts of the DNA fragments to beligated. Unless otherwise stated, transformation was performed asdescribed in the method of Graham, F. and Van der Eb, A., Virology,52:456-457 (1973).

EXAMPLES Example 1 Bacterial Expression and Purification of NAF-1

The DNA sequence encoding NAF-1, ATCC #97343, is initially amplifiedusing PCR oligonucleotide primers corresponding to the 5′ sequences ofthe processed NAF-1 protein (minus the signal peptide sequence) and thevector sequences 3′ to the NAF-1 gene. Additional nucleotidescorresponding to NAF-1 are added to the 5′ and 3′ sequencesrespectively. The 5′ oligonucleotide primer has the sequence 5′GCCATACGGGATCCCAGCCTCTTGGGGGAGAGTCC 3′ (SEQ ID NO:3) contains a BamHIrestriction enzyme site followed by 21 nucleotides of NAF-1 codingsequence starting from the presumed terminal amino acid of the processedprotein codon. The 3′ sequence 5′ GGCATACGTCTAGATTAGACGCAGTTATCAGGGAC 3′(SEQ ID NO:4) contains complementary sequences to an XbaI site and isfollowed by 21 nucleotides of NAF-1. The restriction enzyme sitescorrespond to the restriction enzyme sites on the bacterial expressionvector pQE-9 (Qiagen, Inc. Chatsworth, Calif.). pQE-9 encodes antibioticresistance (Amp^(r)), a bacterial origin of replication (ori), anIPTG-regulatable promoter operator (P/O), a ribosome binding site (RBS),a 6-His tag and restriction enzyme sites. pQE-9 is then digested withBamHI and XbaI. The amplified sequences are ligated into pQE-9 and areinserted in frame with the sequence encoding for the histidine tag andthe RBS. The ligation mixture is then used to transform E. coli strainM15/rep 4 (Qiagen, Inc.) by the procedure described in Sambrook, J. etal., Molecular Cloning: A Laboratory Manual, Cold Spring LaboratoryPress, (1989). M15/rep4 contains multiple copies of the plasmid pREP4,which expresses the lacI repressor and also confers kanamycin resistance(Kan^(r)). Transformants are identified by their ability to grow on LBplates and ampicillin/kanamycin resistant colonies are selected. PlasmidDNA is isolated and confirmed by restriction analysis. Clones containingthe desired constructs are grown overnight (O/N) in liquid culture in LBmedia supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/Nculture is used to inoculate a large culture at a ratio of 1:100 to1:250. The cells are grown to an optical density 600 (O.D.⁶⁰⁰) ofbetween 0.4 and 0.6. IPTG (“Isopropyl-B-D-thiogalacto pyranoside”) isthen added to a final concentration of 1 mM. IPTG induces byinactivating the lacI repressor, clearing the P/O leading to increasedgene expression. Cells are grown an extra 3 to 4 hours. Cells are thenharvested by centrifugation. The cell pellet is solubilized in thechaotropic agent 6 Molar Guanidine HCl. After clarification, solubilizedNAF-1 is purified from this solution by chromatography on aNickel-Chelate column under conditions that allow for tight binding byproteins containing the 6-His tag. NAF-1 is eluted from the column in 6molar guanidine HCl pH 5.0 and for the purpose of renaturation adjustedto 3 molar guanidine HCl, 100 mM sodium phosphate, 10 mmolar glutathione(reduced) and 2 mmolar glutathione (oxidized). After incubation in thissolution for 12 hours the protein is dialyzed to 10 mmolar sodiumphosphate.

Example 2

Cloning and Expression of NAF-1 Using the Baculovirus Expression System

The DNA sequence encoding the full length NAF-1 protein, ATCC #97343,was amplified using PCR oligonucleotide primers corresponding to the 5′and 3′ sequences of the gene:

The 5′ primer has the sequence 5′ GCCATACGGGATCCGCCATCATGGAAAACCCCAGCCCGGCC 3′ (SEQ ID NO:5) and contains a BamHIrestriction enzyme site (in bold) followed by 8 nucleotides resemblingan efficient signal for the initiation of translation in eukaryoticcells (Kozak, M., J. Mol. Biol., 196:947-950 (1987) which is just behindthe first 21 nucleotides of the NAF-1 gene (the initiation codon fortranslation “ATG” is underlined).

The 3′ primer has the sequence 5′ GGCATACGTCTAGATTAGACGCAGTTATCAGGGAC 3′(SEQ ID NO:6) and contains the cleavage site for the restrictionendonuclease XbaI and 21 nucleotides complementary to the 3′ end of thetranslated sequence of the NAF-1 gene. The amplified sequences wereisolated from a 1% agarose gel using a commercially available kit(“Geneclean,” BTO 101 Inc. La Jolla, Calif.). The fragment was thendigested with the endonucleases BamHI and XbaI and then purified againon a 1% agarose gel. This fragment is designated F2.

The vector pRG1 (modification of pVL941 vector, discussed below) is usedfor the expression of the NAF-1 protein using the baculovirus expressionsystem (for review see: Summers, M. D. and Smith, G. E. 1987, A manualof methods for baculovirus vectors and insect cell culture procedures,Texas Agricultural Experimental Station Bulletin No. 1555). Thisexpression vector contains the strong polyhedrin promoter of theAutographa califonica nuclear polyhedrosis virus (AcMNPV) followed bythe recognition sites for the restriction endonucleases BamHI and XbaI.The polyadenylation site of the simian virus (SV)40 is used forefficient polyadenylation. For an easy selection of recombinant virusthe beta-galactosidase gene from E.coli is inserted in the sameorientation as the polyhedrin promoter followed by the polyadenylationsignal of the polyhedrin gene. The polyhedrin sequences are flanked atboth sides by viral sequences for the cell-mediated homologousrecombination of co-transfected wild-type viral DNA. Many otherbaculovirus vectors could be used in place of pRG1 such as pAc373,pVL941 and pAcIM1 (Luckow, V. A. and Summers, M. D., Virology,170:31-39).

The plasmid was digested with the restriction enzymes BamHI and XbaI andthen dephosphorylated using calf intestinal phosphatase by proceduresknown in the art. The DNA was then isolated from a 1% agarose gel usingthe commercially available kit (“Geneclean” BIO 101 Inc., La Jolla,Calif.). This vector DNA is designated V2.

Fragment F2 and the dephosphorylated plasmid V2 were ligated with T4 DNAligase. E.coli XL1 blue cells were then transformed and bacteriaidentified that contained the plasmid (pBacNAF-1) with the NAF-1 geneusing the enzymes BamHI and XbaI. The sequence of the cloned fragmentwas confirmed by DNA sequencing.

5 mg of the plasmid pBacNAF-1 was co-transfected with 1.0 mg of acommercially available linearized baculovirus (“BaculoGold' baculovirusDNA”, Pharmingen, San Diego, Calif.) using the lipofection method(Felgner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)).

1 mg of BaculoGold' virus DNA and 5 mg of the plasmid pBacNAF-1 weremixed in a sterile well of a microtiter plate containing 50 ml of serumfree Grace's medium (Life Technologies Inc., Gaithersburg, Md.).Afterwards 10 ml Lipofectin plus 90 ml Grace's medium were added, mixedand incubated for 15 minutes at room temperature. Then the transfectionmixture was added drop-wise to the Sf9 insect cells (ATCC CRL 1711)seeded in a 35 mm tissue culture plate with 1 ml if Grace's mediumwithout serum. The plate was rocked back and forth to mix the newlyadded solution. The plate was then incubated for 5 hours at 27∞ C. After5 hours the transfection solution was removed from the plate and 1 ml ofGrace's insect medium supplemented with 10% fetal calf serum was added.The plate was put back into an incubator and cultivation continued at27∞ C. for four days.

After four days the supernatant was collected and a plaque assayperformed similar as described by Summers and Smith (supra). As amodification an agarose gel with “Blue Gal” (Life Technologies Inc.,Gaithersburg) was used which allows an easy isolation of blue stainedplaques. (A detailed description of a “plaque assay” can also be foundin the user's guide for insect cell culture and baculovirologydistributed by Life Technologies Inc., Gaithersburg, page 9-10).

Four days after the serial dilution the virus was added to the cells andblue stained plaques were picked with the tip of an Eppendorf pipette.The agar containing the recombinant viruses was then resuspended in anEppendorf tube containing 200 ml of Grace's medium. The agar was removedby a brief centrifugation and the supernatant containing the recombinantbaculovirus was used to infect Sf9 cells seeded in 35 mm dishes. Fourdays later the supernatants of these culture dishes were harvested andthen stored at 4∞ C.

Sf9 cells were grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells were infected with the recombinantbaculovirus V-NAF-1 at a multiplicity of infection (MOI) of 2. Six hourslater the medium was removed and replaced with SF900 II medium minusmethionine and cysteine (Life Technologies Inc., Gaithersburg). 42 hourslater 5 mCi of ³⁵S-methionine and 5 mCi ³⁵S cysteine (Amersham) wereadded. The cells were further incubated for 16 hours before they wereharvested by centrifugation and the labelled proteins visualized bySDS-PAGE and autoradiography.

Example 3

Expression of Recombinant NAF-1 in COS Cells

Expression of plasmid, NAF-1 HA is derived from a vector pcDNAI/Amp(Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillinresistance gene, 3) E.coli replication origin, 4) CMV promoter followedby a polylinker region, an SV40 intron and polyadenylation site. A DNAfragment encoding the entire NAF-1 precursor and a HA tag fused in frameto its 3′ end is cloned into the polylinker region of the vector,placing the recombinant protein expression under control of the CMVpromoter. The HA tag corresponds to an epitope derived from theinfluenza hemagglutinin protein as previously described (I. Wilson, H.Niman, R. Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell37:767, (1984)). The fusion of HA tag to the NAF-1 protein allows easydetection of the recombinant protein with an antibody that recognizesthe HA epitope.

The plasmid construction strategy is described as follows:

The DNA sequence encoding NAF-1; ATCC #97343, is constructed by PCRusing two primers as described in the above examples. The 5′ primercontains a convenient restriction site followed by a portion of NAF-1coding sequence starting from the initiation codon; the 3′ sequencecontains complementary sequences to a convenient restriction site,translation stop codon, HA tag and the last several nucleotides of theNAF-1 coding sequence (not including the stop codon). Therefore, the PCRproduct contains a convenient 5′ and 3′ restriction sites, NAF-1 codingsequence followed by HA tag fused in frame, and a translationtermination stop codon next to the HA tag. The PCR amplified DNAfragment and the vector, pcDNAI/Amp, are digested and ligated. Theligation mixture is transformed into E. coli strain SURE (available fromStratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla,Calif. 92037) the transformed culture is plated on ampicillin mediaplates and resistant colonies are selected. Plasmid DNA is isolated fromtransformants and examined by restriction analysis for the presence ofthe correct fragment. For expression of the recombinant NAF-1, COS cellsare transfected with the expression vector by DEAE-DEXTRAN method (J.Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A LaboratoryManual, Cold Spring Laboratory Press, (1989)). The expression of theNAF-1 HA protein is detected by radiolabelling and immunoprecipitationmethod (E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory Press, (1988)). Cells are labelled for 8 hours with³⁵S-cysteine two days post transfection. Culture media is then collectedand cells were lysed with detergent (RIPA buffer (150 mM NaCl, 1% NP-40,0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM Tris, pH 7.5) (Wilson, I. et al.,Id. 37:767 (1984)). Both cell lysate and culture media are precipitatedwith an HA specific monoclonal antibody. Proteins precipitated areanalyzed on 15% SDS-PAGE gels.

Example 4

Expression via Gene Therapy

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in tissue-culture medium and separated into smallpieces. Small chunks of the tissue are placed on a wet surface of atissue culture flask, approximately ten pieces are placed in each flask.The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ram's F12 media, with 10% FBS, penicillinand streptomycin, is added. This is then incubated at 37∞ C. forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerge. The monolayer istrypsinized and scaled into larger flasks.

pMV-7 (Kirschmeicr, P. T. et al, DNA, 7:219-25 (1988) flanked by thelong terminal repeats of the Moloney murine sarcoma virus, is digestedwith EcoRI and HindIII and subsequently treated with calf intestinalphosphatase. The linear vector is fractionated on agarose gel andpurified, using glass beads.

The cDNA encoding a polypeptide of the present invention is amplifiedusing PCR primers which correspond to the 5′ and 3′ end sequencesrespectively. The 5′ primer containing an EcoRI site and the 3′ primerfurther includes a HindIII site. Equal quantities of the Moloney murinesarcoma virus linear backbone and the amplified EcoRI and HindIIIfragment are added together, in the presence of T4 DNA ligase. Theresulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is used to transformbacteria HB101, which are then plated onto agar-containing kanamycin forthe purpose of confirming that the vector had the gene of interestproperly inserted.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissueculture to confluent density in Dulbecco's Modified Eagles Medium (DMEM)with 10% calf serum (CS), penicillin and streptomycin. The MSV vectorcontaining the gene is then added to the media and the packaging cellsare transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging cells arenow referred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently,the media is harvested from a 10 cm plate of confluent producer cells.The spent media, containing the infectious viral particles, is filteredthrough a millipore filter to remove detached producer cells and thismedia is then used to infect fibroblast cells. Media is removed from asub-confluent plate of fibroblasts and quickly replaced with the mediafrom the producer cells. This media is removed and replaced with freshmedia. If the titer of virus is high, then virtually all fibroblastswill be infected and no selection is required. If the titer is very low,then it is necessary to use a retroviral vector that has a selectablemarker, such as neo or his.

The engineered fibroblasts are then injected into the host, either aloneor after having been grown to confluence on cytodex 3 microcarrierbeads. The fibroblasts now produce the protein product.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

19 1 1105 DNA homo sapiens CDS (19)..(1011) 1 cgctgctcct gccgggtg atggaa aac ccc agc ccg gcc gcc gcc ctg ggc 51 Met Glu Asn Pro Ser Pro AlaAla Ala Leu Gly 1 5 10 aag gcc ctc tgc gct ctc ctc ctg gcc act ctc ggcgcc gcc ggc cag 99 Lys Ala Leu Cys Ala Leu Leu Leu Ala Thr Leu Gly AlaAla Gly Gln 15 20 25 cct ctt ggg gga gag tcc atc tgt tcc gcc aga gcc ctggcc aaa tac 147 Pro Leu Gly Gly Glu Ser Ile Cys Ser Ala Arg Ala Leu AlaLys Tyr 30 35 40 agc atc acc ttc acg ggc aag tgg agc cag acg gcc ttc cccaag cag 195 Ser Ile Thr Phe Thr Gly Lys Trp Ser Gln Thr Ala Phe Pro LysGln 45 50 55 tac ccc ctg ttc cgc ccc cct gcc cag tgg tct tcg ctg ctg ggggcc 243 Tyr Pro Leu Phe Arg Pro Pro Ala Gln Trp Ser Ser Leu Leu Gly Ala60 65 70 75 gcg cat agc tcc gac tac agc atg tgg agg aag aac cag tac gtcagt 291 Ala His Ser Ser Asp Tyr Ser Met Trp Arg Lys Asn Gln Tyr Val Ser80 85 90 aac ggg ctg cgc gac ttt gcg gag cgc ggc gag gcc tgg gcg ctg atg339 Asn Gly Leu Arg Asp Phe Ala Glu Arg Gly Glu Ala Trp Ala Leu Met 95100 105 aag gag atc gag gcg gcg ggg gag gcg ctg cag agc gtg cac gcg gtg387 Lys Glu Ile Glu Ala Ala Gly Glu Ala Leu Gln Ser Val His Ala Val 110115 120 ttt tcg gcg ccc gcc gtc ccc agc ggc acc ggg cag acg tcg gcg gag435 Phe Ser Ala Pro Ala Val Pro Ser Gly Thr Gly Gln Thr Ser Ala Glu 125130 135 ctg gag gtg cag cgc agg cac tcg ctg gtc tcg ttt gtg gtg cgc atc483 Leu Glu Val Gln Arg Arg His Ser Leu Val Ser Phe Val Val Arg Ile 140145 150 155 gtg ccc agc ccc gac tgg ttc gtg ggc gtg gac agc ctg gac ctgtgc 531 Val Pro Ser Pro Asp Trp Phe Val Gly Val Asp Ser Leu Asp Leu Cys160 165 170 gac ggg gac cgt tgg cgg gaa cag gcg gcg ctg gac ctg tac ccctac 579 Asp Gly Asp Arg Trp Arg Glu Gln Ala Ala Leu Asp Leu Tyr Pro Tyr175 180 185 gac gcc ggg acg gac agc ggc ttc acc ttc tcc tcc ccc aac ttcgcc 627 Asp Ala Gly Thr Asp Ser Gly Phe Thr Phe Ser Ser Pro Asn Phe Ala190 195 200 acc atc ccg cag gac acg gtg acc gag ata acg tcc tcc tct cccagc 675 Thr Ile Pro Gln Asp Thr Val Thr Glu Ile Thr Ser Ser Ser Pro Ser205 210 215 cac ccg gcc aac tcc ttc tac tac ccg cgg ctg aag gcc ctg cctccc 723 His Pro Ala Asn Ser Phe Tyr Tyr Pro Arg Leu Lys Ala Leu Pro Pro220 225 230 235 atc gcc agg gtg aca ctg gtg cgg ctg cga cag agc ccc agggcc ttc 771 Ile Ala Arg Val Thr Leu Val Arg Leu Arg Gln Ser Pro Arg AlaPhe 240 245 250 atc cct ccc gcc cca gtc ctg ccc agc agg gac aat gag attgta gac 819 Ile Pro Pro Ala Pro Val Leu Pro Ser Arg Asp Asn Glu Ile ValAsp 255 260 265 agc gcc tca gtt cca gaa acg ccg ctg gac tgc gag gtc tccctg tgg 867 Ser Ala Ser Val Pro Glu Thr Pro Leu Asp Cys Glu Val Ser LeuTrp 270 275 280 tcg tcc tgg gga ctg tgc gga ggc cac tgt ggg agg ctc gggacc aag 915 Ser Ser Trp Gly Leu Cys Gly Gly His Cys Gly Arg Leu Gly ThrLys 285 290 295 agc agg act cgc tac gtc cgg gtc cag ccc gcc aac aac gggagc ccc 963 Ser Arg Thr Arg Tyr Val Arg Val Gln Pro Ala Asn Asn Gly SerPro 300 305 310 315 tgc ccc gag ctc gaa gaa gag gct gag tgc gtc cct gataac tgc gtc 1011 Cys Pro Glu Leu Glu Glu Glu Ala Glu Cys Val Pro Asp AsnCys Val 320 325 330 taagaccaga gccccgcagc ccctggggcc ccccggagccatggggtgtc gggggctcct 1071 gtgcaggctc atgctgcagg cggccgaggg caca 1105 2331 PRT homo sapiens 2 Met Glu Asn Pro Ser Pro Ala Ala Ala Leu Gly LysAla Leu Cys Ala 1 5 10 15 Leu Leu Leu Ala Thr Leu Gly Ala Ala Gly GlnPro Leu Gly Gly Glu 20 25 30 Ser Ile Cys Ser Ala Arg Ala Leu Ala Lys TyrSer Ile Thr Phe Thr 35 40 45 Gly Lys Trp Ser Gln Thr Ala Phe Pro Lys GlnTyr Pro Leu Phe Arg 50 55 60 Pro Pro Ala Gln Trp Ser Ser Leu Leu Gly AlaAla His Ser Ser Asp 65 70 75 80 Tyr Ser Met Trp Arg Lys Asn Gln Tyr ValSer Asn Gly Leu Arg Asp 85 90 95 Phe Ala Glu Arg Gly Glu Ala Trp Ala LeuMet Lys Glu Ile Glu Ala 100 105 110 Ala Gly Glu Ala Leu Gln Ser Val HisAla Val Phe Ser Ala Pro Ala 115 120 125 Val Pro Ser Gly Thr Gly Gln ThrSer Ala Glu Leu Glu Val Gln Arg 130 135 140 Arg His Ser Leu Val Ser PheVal Val Arg Ile Val Pro Ser Pro Asp 145 150 155 160 Trp Phe Val Gly ValAsp Ser Leu Asp Leu Cys Asp Gly Asp Arg Trp 165 170 175 Arg Glu Gln AlaAla Leu Asp Leu Tyr Pro Tyr Asp Ala Gly Thr Asp 180 185 190 Ser Gly PheThr Phe Ser Ser Pro Asn Phe Ala Thr Ile Pro Gln Asp 195 200 205 Thr ValThr Glu Ile Thr Ser Ser Ser Pro Ser His Pro Ala Asn Ser 210 215 220 PheTyr Tyr Pro Arg Leu Lys Ala Leu Pro Pro Ile Ala Arg Val Thr 225 230 235240 Leu Val Arg Leu Arg Gln Ser Pro Arg Ala Phe Ile Pro Pro Ala Pro 245250 255 Val Leu Pro Ser Arg Asp Asn Glu Ile Val Asp Ser Ala Ser Val Pro260 265 270 Glu Thr Pro Leu Asp Cys Glu Val Ser Leu Trp Ser Ser Trp GlyLeu 275 280 285 Cys Gly Gly His Cys Gly Arg Leu Gly Thr Lys Ser Arg ThrArg Tyr 290 295 300 Val Arg Val Gln Pro Ala Asn Asn Gly Ser Pro Cys ProGlu Leu Glu 305 310 315 320 Glu Glu Ala Glu Cys Val Pro Asp Asn Cys Val325 330 3 36 DNA oligonucleotide primer_bind (1)..(36) 5′ primercontaining a BamHI restriction enzyme site followed by 21 nucleotides ofNAD-1 coding sequence. 3 gccatacggg atccccagcc tcttggggga gagtcc 36 4 35DNA oligonucleotide primer_bind (1)..(35) 3′ primer containingcomplementary sequence to an XbaI site followed by 21 nucleotides ofNAF-1 sequence. 4 ggcatacgtc tagattagac gcagttatca gggac 35 5 41 DNAoligonucleotide primer_bind (1)..(41) 5′ primer containing a BamHIrestriction enzyme site followed by 8 nucleotides resembling anefficient signal for initiation of translation in eukaryotic cellsfollowed by 21 nucleotides of NAF-1 sequence. 5 gccatacggg atccgccatcatggaaaacc ccagcccggc c 41 6 35 DNA oligonucleotide primer_bind(1)..(35) 3′ primer containing the cleavage site for XbaI restrictionendonuclease and 21 nucleotides complementary to the 3′ end of thetranslated sequence of the NAF-1 gene. 6 ggcatacgtc tagattagacgcagttatca gggac 35 7 392 PRT rat 7 Pro Thr Gly Thr Gly Cys Val Ile LeuLys Ala Ser Ile Val Gln Lys 1 5 10 15 Arg Ile Ile Tyr Phe Gln Asp GluGly Ser Leu Thr Lys Lys Leu Cys 20 25 30 Glu Gln Asp Pro Thr Leu Asp GlyVal Thr Asp Arg Pro Ile Leu Asp 35 40 45 Cys Cys Ala Cys Gly Thr Ala LysTyr Arg Leu Thr Phe Tyr Gly Asn 50 55 60 Trp Ser Glu Lys Thr His Pro LysAsp Tyr Pro Arg Arg Ala Asn His 65 70 75 80 Trp Ser Ala Ile Ile Gly GlySer His Ser Lys Asn Tyr Val Leu Trp 85 90 95 Glu Tyr Gly Gly Tyr Ala SerGlu Gly Val Lys Gln Val Ala Glu Leu 100 105 110 Gly Ser Pro Val Lys MetGlu Glu Glu Ile Arg Gln Gln Ser Asp Glu 115 120 125 Val Leu Thr Val IleLys Ala Lys Ala Gln Trp Pro Ser Trp Gln Pro 130 135 140 Val Asn Val ArgAla Ala Pro Ser Ala Glu Phe Ser Val Asp Arg Thr 145 150 155 160 Arg HisLeu Met Ser Phe Leu Thr Met Met Gly Pro Ser Pro Asp Trp 165 170 175 AsnVal Gly Leu Ser Ala Glu Asp Leu Cys Thr Lys Glu Cys Gly Trp 180 185 190Val Gln Lys Val Val Gln Asp Leu Ile Pro Trp Asp Ala Gly Thr Asp 195 200205 Ser Gly Val Thr Tyr Glu Ser Pro Asn Lys Pro Thr Ile Pro Gln Glu 210215 220 Lys Ile Arg Pro Leu Thr Ser Leu Asp His Pro Gln Ser Pro Phe Tyr225 230 235 240 Asp Pro Glu Gly Gly Ser Ile Thr Gln Val Ala Arg Val ValIle Glu 245 250 255 Arg Ile Ala Arg Lys Gly Glu Gln Cys Asn Ile Val ProAsp Asn Val 260 265 270 Asp Asp Ile Val Ala Asp Leu Ala Pro Glu Glu LysAsp Glu Asp Asp 275 280 285 Thr Pro Glu Thr Cys Ile Tyr Ser Asn Trp SerPro Trp Ser Ala Cys 290 295 300 Ser Ser Ser Thr Cys Glu Lys Gly Lys ArgMet Arg Gln Arg Met Leu 305 310 315 320 Lys Ala Gln Leu Asp Leu Ser ValPro Cys Pro Asp Thr Gln Asp Phe 325 330 335 Gln Pro Cys Met Gly Pro GlyCys Ser Asp Glu Asp Gly Ser Thr Cys 340 345 350 Thr Met Ser Glu Trp IleThr Trp Ser Pro Cys Ser Val Ser Cys Gly 355 360 365 Met Gly Met Arg SerArg Glu Arg Tyr Val Lys Gln Phe Pro Glu Asp 370 375 380 Gly Ser Val CysMet Leu Pro Thr 385 390 8 52 PRT rat 8 Cys Ile Tyr Ser Asn Trp Ser ProTrp Ser Ala Cys Ser Ser Ser Thr 1 5 10 15 Cys Glu Lys Gly Lys Arg MetArg Gln Arg Met Leu Lys Ala Gln Leu 20 25 30 Asp Leu Ser Val Pro Cys ProAsp Thr Gln Asp Phe Gln Pro Cys Met 35 40 45 Gly Pro Gly Cys 50 9 53 PRTrat 9 Cys Thr Met Ser Glu Trp Ile Thr Trp Ser Pro Cys Ser Val Ser Cys 15 10 15 Gly Met Gly Met Arg Ser Arg Glu Arg Tyr Val Lys Gln Phe Pro Glu20 25 30 Asp Gly Ser Val Cys Met Leu Pro Thr Glu Glu Thr Glu Lys Cys Thr35 40 45 Val Asn Glu Glu Cys 50 10 52 PRT rat 10 Cys Leu Val Thr Glu TrpGly Glu Trp Asp Asp Cys Ser Ala Thr Cys 1 5 10 15 Gly Met Gly Met LysLys Arg His Arg Met Val Lys Met Ser Pro Ala 20 25 30 Asp Gly Ser Met CysLys Ala Glu Thr Ser Gln Ala Glu Lys Cys Met 35 40 45 Met Pro Glu Cys 5011 51 PRT rat 11 Cys Leu Leu Ser Pro Trp Ser Glu Trp Ser Asp Cys Ser ValThr Cys 1 5 10 15 Gly Lys Gly Met Arg Thr Arg Gln Arg Met Leu Lys SerLeu Ala Glu 20 25 30 Leu Gly Asp Cys Asn Glu Asp Leu Glu Gln Ala Glu LysCys Met Leu 35 40 45 Pro Glu Cys 50 12 52 PRT rat 12 Cys Glu Leu Ser GluTrp Ser Gln Trp Ser Glu Cys Asn Lys Ser Cys 1 5 10 15 Gly Lys Gly HisMet Ile Arg Thr Arg Thr Ile Gln Met Glu Pro Gln 20 25 30 Phe Gly Gly AlaPro Cys Pro Glu Thr Val Gln Arg Lys Lys Cys Arg 35 40 45 Ala Arg Lys Cys50 13 53 PRT rat 13 Cys Arg Met Arg Pro Trp Thr Ala Trp Ser Glu Cys ThrLys Leu Cys 1 5 10 15 Gly Gly Gly Ile Gln Glu Arg Tyr Met Thr Val LysLys Arg Phe Lys 20 25 30 Ser Ser Gln Phe Thr Ser Cys Lys Asp Lys Lys GluIle Arg Ala Cys 35 40 45 Asn Val His Pro Cys 50 14 50 PRT Homo sapiens14 Cys Leu Val Ser Glu Trp Ser Glu Trp Ser Asp Cys Ser Thr Cys Gly 1 510 15 Lys Gly Met Arg Ser Arg Thr Arg Met Val Lys Met Ser Pro Ala Asp 2025 30 Gly Ser Pro Cys Pro Asp Thr Glu Glu Ala Glu Lys Cys Met Val Pro 3540 45 Glu Cys 50 15 15 000 16 506 DNA homo sapiens misc_feature(11)..(11) n is equal to a, t, c, or g 16 gaattcggca naggnnaaaccccagcccgg ctgccgccct gggcaaggcc tnctgcgctc 60 tcctcctggc cactctcggcgccggcacca gcctcttggg ggagagtcca tctnttccgc 120 cagagccccg gccaaatacagcatcacctt cacgggcaag tggagccaga cggccttccc 180 caagcagtac cccctgttccgcccccctgc gcatggtntt cgctgctggg ggccgcgcat 240 agctccgact acagcatgtggaggaagaac cagtacgtca taaacgggct gcgcgacttt 300 ncggagcggc gaggcctnggncgttgatga aggagatccg ggnggcgggg gaggcgtnca 360 anaggtgnca agagttnttttcggggcccg gttccccaan ggnaacnggn aaacgttggg 420 ggntttnnag tttnaagaagnaattnttgg tttttttttg ggtgggattt tnccaacccn 480 attgtttntg ggntggaaaattngac 506 17 316 DNA homo sapiens misc_feature (5)..(5) n is equal toa, t, c, or g 17 ggcanngcca gtacgtcata acgggctgcg cgactttgcg gangcggcgaggcctgggcg 60 ctgatgaagg agatcaaggc ggcgggggag gcgctgcaga ggtgcacgaggtgttttcgg 120 cgcccggtnn cccagcgnca ccnggcagac gtcggcgaac tggnaggtgcagcgcaggca 180 ctcgctggtc tcgtttgtgg tgcgcatcgt gcccagcccc gactggttcgtgggcgtgga 240 cagcctggga cctgtganaa cggggacctt tngcgngnaa caggcgncgttggacctgta 300 nccctacgac gncggg 316 18 316 DNA homo sapiensmisc_feature (5)..(5) n is equal to a, t, c, or g 18 ggcanngccagtacgtcata acgggctgcg cgactttgcg gangcggcga ggcctgggcg 60 ctgatgaaggagatcaaggc ggcgggggag gcgctgcaga ggtgcacgag gtgttttcgg 120 cgcccggtnncccagcgnca ccnggcagac gtcggcgaac tggnaggtgc agcgcaggca 180 ctcgctggtctcgtttgtgg tgcgcatcgt gcccagcccc gactggttcg tgggcgtgga 240 cagcctgggacctgtganaa cggggacctt tngcgngnaa caggcgncgt tggacctgta 300 nccctacgacgncggg 316 19 53 PRT homo sapiens 19 Cys Glu Val Ser Leu Trp Ser Ser TrpGly Leu Cys Gly Gly His Cys 1 5 10 15 Gly Arg Leu Gly Thr Lys Ser ArgThr Arg Tyr Val Arg Val Gln Pro 20 25 30 Ala Asn Asn Gly Ser Pro Cys ProGlu Leu Glu Glu Glu Ala Glu Cys 35 40 45 Val Pro Asp Asn Cys 50

What is claimed is:
 1. An isolated polypeptide comprising an amino acidsequence selected from the group consisting of: (a) amino acids 1 to 331of SEQ ID NO:2; (b) the complete amino acid sequence encoded by the cDNAclone contained in ATCC Deposit No. 97343; (c) amino acids 2 to 331 ofSEQ ID NO:2; (d) the complete amino acid sequence excepting theN-terminal methionine encoded by the cDNA clone contained in ATCCDeposit No. 97343; (e) amino acids 24 to 331 of SEQ ID NO:2; (f) aminoacids 27 to 331 of SEQ ID NO:2; and (g) the amino acid sequence of themature form of NAF-1 encoded by the cDNA clone contained in ATCC DepositNo.
 97343. 2. The isolated polypeptide of claim 1 wherein said aminoacid sequence is (a).
 3. The isolated polypeptide of claim 1 whereinsaid amino acid sequence is (b).
 4. The isolated polypeptide of claim 1wherein said amino acid sequence is (c).
 5. The isolated polypeptide ofclaim 1 wherein said amino acid sequence is (d).
 6. The isolatedpolypeptide of claim 1 wherein said amino acid sequence is (e).
 7. Theisolated polypeptide of claim 1 wherein said amino acid sequence is (f).8. The isolated polypeptide of claim 1 wherein said amino acid sequenceis (g).
 9. An epitope bearing fragment of the human NAF-1 polypeptideselected from the group consisting of: (a) an epitope bearing fragmentconsisting of at least amino acids 75 to 100 of SEQ ID NO:2; (b) anepitope bearing fragment consisting of at least amino acids 168 to 180of SEQ ID NO:2; (c) an epitope bearing fragment consisting of at leastamino acids 204 to 226 of SEQ ID NO:2; (d) an epitope bearing fragmentconsisting of at least amino acids 258 to 281 of SEQ ID NO:2; and, (e)an epitope bearing fragment consisting of at least amino acids 291 to327 of SEQ ID NO:2.
 10. The epitope bearing fragment of claim 9 whereinsaid fragment is (a).
 11. The epitope bearing fragment of claim 9wherein said fragment is (b).
 12. The epitope bearing fragment of claim9 wherein said fragment is (c).
 13. The epitope bearing fragment ofclaim 9 wherein said fragment is (d).
 14. The epitope bearing fragmentof claim 9 wherein said fragment is (e).